The use of gel electrophoresis is currently the ubiquitous technique for the separation of biological materials. Typical applications include separation of nucleic acid fragments of various sizes either in the context of sequence determination, detection of polymorphisms, or verification of sizes in other contexts. Other frequently used applications include separation of proteins, verification of homogeneity or purity, identification of post-translational modifications, and confirmation of molecular weight.
In all of these procedures, mixed samples of proteins or nucleic acids are applied to electrophoretic gels and the components separated by application of an electric field across the gel. Regardless of the manner in which the gel is developed, the resulting pattern of migration of the proteins or nucleic acids contained in the sample must be detected in some manner.
To conduct this detection for proteins, the gel support is typically contacted with a blotting membrane to which the entities are transferred as they appear on the gel. The “spots” are then detected, at a minimum, by blocking the membrane with a protein or detergent solution to reduce non-specific binding. The biological entity is then incubated with an antibody specific for the antigen on the membrane. The membrane is then extensively washed to remove any contaminants, unbound blocking proteins, and antibodies. Next, the membrane is treated and incubated with a secondary enzyme-, radioisotope-, fluorophore-, or biotin-conjugated antibody specific for the primary antibody. The membrane is again extensively washed to remove any unbound secondary antibody. A detection reagent, generally a chromogenic, chemiluminescent, fluorescent, radiological, or streptavidin-labeled material, is then applied which either binds to, or is a substrate of the enzyme-conjugate. Lastly, the appropriate detection device is used to determine the presence, absence, position, quantity, etc. of the biological entity.
Furthermore, in order to detect biological entities within the context of an organ, tissue, cell, etc., current immunohistochemical techniques use a process similar to that generally outlined above. Typical applications include identification of biological entities or their location in histological sections or preparations of tissues, organs cells, organisms etc., or verification of biological entities in other contexts.
In these procedures, histological sections or preparations are applied to glass slides. Regardless of the manner in which the histological sections or preparations are applied to glass slide, the resulting pattern, identification, location, etc. of the biological entities contained in the sample must be detected in some manner.
To conduct this detection, the biological entity on the glass slide is incubated with an antibody specific for the antigen on the histological preparation. The glass slide is then extensively washed to remove any contaminants, unbound blocking proteins, and antibodies. Next, the histological preparation/glass slide is treated and incubated with a secondary enzyme-, radioisotope-, fluorophore-, or biotin-conjugated antibody specific for the primary antibody. The membrane is again extensively washed to remove any unbound secondary antibody. A detection reagent, generally a chromogenic, chemiluminescent, fluorescent, radiological, or streptavidin-labeled material, is then applied which either binds to, or is a substrate of the enzyme-conjugate. Lastly, the appropriate detection device is used to determine the presence, absence, position, quantity, etc. of the biological entity.
The last several steps of these processes generally take from 3-6 hours to overnight, depending on the speed of the reaction between the selected reagents, the membrane composition, and the biological entity. The process requires multiple incubation periods of the membrane on a rocking or other suitable mixing platform. It is a lengthy process that most researchers dislike and which consumes a large volume of reagents.